Rice Vinegar Residue Reduces Adipocyte Size and Hyperlipidemia by Inhibiting Lipids Absorption in Hamsters

TONG Litao, JU Zhiyuan, GENG Donghui, WANG Lili, ZHOU Xianrong, ZHOU Sumei

(Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences, Key Laboratory of Agro-products Processing,Ministry of Agriculture and Rural Affairs, Beijing 100193, China)

Abstract: Rice vinegar residue, a by-product of rice vinegar making, is wasted in enormous quantity, which has been believed to have potential effects in the regulation of lipid metabolism. This study aimed to investigate the effects of rice vinegar residue on lipid metabolism in hypercholesterolemic hamsters. Rice vinegar residue at doses of 200 and 500 mg/(kg mb·d) and rice vinegar at 200 mg/(kg mb·d) were separately given by gavage to hamsters for 30 days. Both rice vinegar residue and rice vinegar significantly lowered the concentration of low-density lipoprotein cholesterol in plasma (P < 0.05), reduced the average size of adipocytes in white adipose tissues, and increased the fecal concentrations of triglycerides, total cholesterol and bile acids (P < 0.05). Moreover, vinegar residue disrupted solubilization of intestinal cholesterol into micelles (P < 0.05),as well as inhibited its binding to cholate (P < 0.05). On the other hand, vinegar residue had no inhibitory effect on lipase activity. Eventually, we conclude that rice vinegar residue has potential beneficial effects on lipid metabolism and obesity by preventing the absorption of cholesterol in the intestine and promoting the excretion of fecal lipids and cholesterol synthesis in the liver, which may be related to the combination between vinegar residue and cholate.

Keywords: rice vinegar residue; hypercholesterolemia; adipocyte size; fecal lipids; lipid absorption

The rice vinegar residue obtained from brown rice is a by-product of producing rice vinegar. About 1.8-2.2 million tons of rice vinegar residue is dumped annually in China, which is used in process of low value-added products manufacturing, such as animal feed and soilless culture matrix[1-2]. Numerous studies have indicated that rice vinegar shows beneficial effects on improving high fat-induced beta cell dysfunction, protecting liver, reducing body weight, and regulating lipids metabolism[3-5]. A few studies indicated that the bioactivities of rice vinegar residue include regulation of carbohydrate metabolism, promotion of calcium absorption and amelioration of asthmatic bronchial-bronchiolar lesions[6].

Rice vinegar residue is rich in variety of edible components, including rice oil, proteins, and a large amount of dietary fiber. These components have been reported to show the cholesterol-lowering function[7-8]. Moreover, the starch in rice is consumed through fermentation, which yields extensive components in rice vinegar residue such as dietary fiber, oil and proteins, and thus enhances their activities.Previous studies reported that the vinegar residue have 65.5% neutral detergent fiber, 44.5% acid detergent fiber,6.2% crude fat, and 13.9% proteins, including peptides and amino acids[9]. However, whether rice vinegar residue could possibly affect lipids absorption and metabolism or not in hypercholesterolemic hamsters is still unknown.

Besides, the previous study indicated that oral administration of Kurozu (brown rice vinegar) decreased the adipocyte size by reducing peroxisome proliferatorsactivated receptor γ and adipocyte fatty acid binding protein mRNA expression levels and inhibiting lipids absorption in adipocytes[10]. In preventing obesity and metabolic syndrome,reduction in adipocyte size plays a key role, because hypertrophic adipocytes secrete much more tumor necrosis factor and lipids than normal adipocytes do[11]. The rice vinegar can inhibit obesity by reducing dietary fat absorption and adipocyte size. The effects of rice vinegar residue can be employed in multiple dimensions to maximize the outputs.

In the present study, the chemical composition in rice vinegar residue was determined. Rice vinegar, as a well-known hypolipidemic functional food, was used to investigate the effects of orally administrated rice vinegar residue on lipids metabolisms and adipocyte size in hamsters fed with a hypercholesterolemic diet. Fecal lipids excretion,pancreatic lipase activity, micellar solution of cholesterol,binding activity of cholate, and pancreatic lipase activity were determined to clarify the underlying mechanism of the hypercholesterolemic effect.

1 Materials and methods

1.1 Animals, materials and reagents

Male golden hamsters were provided by Vital River Lab Animal Technology Co., Ltd. (Certificate No. SCXK-2004-0001).

Rice vinegar residue and rice vinegar were provided by Beijing Er Shang Group (Beijing, China). The rice vinegar residue was crushed by high-speed crusher and passed through 200 mesh sieve after being dried at 80 ℃ for 10 h.

Tissue total cholesterol (TC) assay kit, tissue triglyceride(TG) assay kit, tissue free cholesterol assay kit, cholesterol assay kit Beijing Applygen Technologies Co. Ltd.;Enzyme-linked immunosorbent assay kit Nanjing Sen Shellfish Gamma Biotechnology Co. Ltd..

1.2 Instruments and equipments

Automatic chemistry analyzer Hitachi group,Tokyo, Japan.

1.3 Methods

1.3.1 Chemical compositions of rice vinegar residue and rice vinegar

AOAC methods were used to determine nitrogen(954.01), lipids (920.39), ash (923.03), crude fibre (962.09),total sugars (968.28), short chain fatty acids (996.06) and moisture (925.09) contents. Xylanase was determined by a method of Englyst et al[12].

1.3.2 Animals and diets

Male golden hamsters (4 weeks, n = 40) were acclimatized in stainless steel cages to the laboratory conditions (temperature was 21-24 ℃, with lights on 08:00-20:00) for 7 days prior to the study. Hamsters were divided into 4 groups (control, low dose of rice vinegar residue (L-RV), high dose of rice vinegar residue(H-RV) and rice vinegar with consistent average body weight among four groups. Diets were prepared according to the American Institute of Nutrition 93G formula with some modifications. The diets contained 397 corn starch, 200 casein, 132 maltodextrin 10, 70 soy bean oil, 50 cellulose,50 lard, 38.486 sucrose, 35 mineral mixture, 10 vitamin mixture, 10 cholesterol, 3 L-cystine, 2.5 choline bitartrate,and 2 bile salt in g/kg. Rice vinegar residue or rice vinegar was orally administered to the hamsters at 9:00 each day for 30 days. Rice vinegar residue dissolved in 0.5% sodium carboxymethyl cellulose (CMC) was administered at a dose of 200 and 500 mg/kg mbto the L-RV and H-RV groups,respectively. Rice vinegar dissolved in 0.5% CMC was administered at a dose of 200 mg/kg mbto the rice vinegar group. Only 0.5% CMC was orally administered to the control group.

The plasma was taken from the eye-veniplex on the 0th, 10thand 20thday of the study, respectively. Hamster feces were collected for 1 week before all hamsters were slaughtered by the removal of blood from the eye-veniplex after being fasted for 5 h. Livers and white adipose tissues(mesenteric, perirenal and epididymal) were frozen by liquid nitrogen and kept at -20 ℃.

All the experiments were approved by the Bioethics Committee of the Institute of Medicinal Plant Development,Chinese Academy of Medical Sciences and Peking Union Medical College.

1.3.3 Growth and metabolic parameters of hamsters

The plasma lipids concentrations were measured by automatic chemistry analyzer. Liver lipids were extracted and the TC, TG, free cholesterol concentrations in liver were measured with kits. Faeces were lyophilized and weighed. The total lipids in feces were measured by Soxhlet method. The fecal cholesterol content and bile acids (BAs) content were measured by cholesterol assay kit and rat BAs kit, respectively.

1.3.4 Morphologic and pathologic analysis of tissue in hamsters

White adipose tissues and liver were rinsed by saline solution, and fixed in 10% neutral formalin buffer solution.After being embedded in paraffin, the tissues were then cut into 10 μm sections and stained with hematoxylin. Adipocyte cell size was compared through counting up numbers of cells in 40 × 10 times micrograph. Liver damage conditions were further assessed by pathologic examination.

1.3.5 Micellar solution of cholesterol

An emulsion (pH 7.4) containing 10 mmol/L sodium taurocholate, 2 mmol/L cholesterol, 5 mmol/L oleic acid,132 mmol/L NaCl, 15 mmol/L sodium chloride, and the samples with various concentrations were prepared by sonication (400 W, 20 kHz, 20 min) and then kept at 37 ℃for 24 h. After centrifugation at 6 000 × g for 30 min,cholesterol concentration of the supernatant was measured with cholesterol kits at λ = 500 nm. Cholesterol concentration in micelles was calculated according to formula (1).

where A0is absorbancy of control group; A1is absorbancy of test group.

1.3.6 Binding activity of cholate

Samples with different concentrations and 4 mL of cholate solution (0.02 mmol/L sodium taurocholate dissolved in 0.1 mol/L phosphate buffer in pH 7.0) were added into 10 mL plug tubes and incubated at 37 ℃ for 1 h. The obtained mixture was centrifuged at 3 000 × g for 20 min, and the supernatant (2.5 mL) of each sample mixed with 60% H2SO4(7.5 mL), which was then heated in 70 ℃ water for 20 min and put in ice bath for 5 min. The concentration of cholate in the samples was determined by absorbance at 387 nm.

1.3.7 Inhibition of pancreatic lipase activity in vitro

An emulsion containing 0.15% nitrophenol palmitate and isopropyl alcohol was formed by ultrasonic treatment.The emulsion was diluted with 50 mmol/L phosphate buffer(containing 0.1% acacia gum and 0.4% casein X-100,pH 7.0) in a ratio of 1:10. The samples with different concentrations were added into 4 mL of the diluted emulsion. After incubating at 37 ℃ for 5 min, 1 mL of 0.5 mg/mL pancreatic lipase was also added into the diluted emulsion, followed by the incubation at 37 ℃ for 15 min,and then 0.4 mL of 0.1 mol/L sodium carbonate solution was added for stopping the reaction. The absorbance of nitro phenol at 410 nm showed the enzymatic activity.The pancreatic lipase activity was calculated according to the formula (2).

where A0is the absorbance of control group; and A1is the value of test group.

1.4 Statistical analysis

The data were expressed as means with standard errors (SE)or standard deviation (SD) and analyzed by Tukey-Kramer’s multiple comparison post hoc test. The analysis was carried out with SPSS (Version 12.0 for Windows). Differences were considered to be significant at P < 0.05.

2 Results and analysis

2.1 Chemical compositions of rice vinegar residue and rice vinegar

As shown in Table 1, rice vinegar residue contained mainly 7.02 g/100 g proteins, 12.79 g/100 g lipids,31.47 g/100 g crude fiber, and 27.37 g/100 g xylanase.

Table 1 Compositions of rice vinegar residue and rice vinegar

2.2 Growth and metabolism parameters of hamsters

Table 2 Effects of orally administered L-VR, H-VR and rice vinegar on growth and metabolic parameters

Growth and metabolism parameters of hamsters were shown in Table 2. There were no differences in body weight, food intake, and feed efficiency among the 4 groups. That means the amount of intake of lipids among groups stay in the same level. Adipose tissue and liver weights were also not affected.

The plasma LDL-C concentrations in the L-VR and H-VR groups were significantly reduced compared with that in the control group (P < 0.05), as well as that in the rice vinegar group. The plasma HDL-C concentrations in the L-VR and H-VR groups were significantly higher than that in the control group (P < 0.05). As for the plasma TG concentration, only the H-VR group led to a significant decrease compared with those of the control group (P < 0.05).There was no effect of L-RV and H-RV groups on plasma TC concentration.

There was no obvious effect of L-RV and H-RV groups on liver cholesterol concentration. The liver TG concentration of L-RV and H-RV groups were obviously lower than those of the control group (P < 0.05), contrary to the rice vinegar group was not. Both rice vinegar residue and rice vinegar increased fecal TC concentration, while only the H-VR group significantly increased fecal BAs concentrations(P < 0.05). Fecal total lipids content in the H-VR group was the highest one among 4 groups (P < 0.05) and that in rice vinegar group was the second highest (P < 0.05),while that of the L-VR group was not different from that of the control group.

2.3 Pathologic analysis of tissue in hamsters

The hepatic sinus dilating and congesting was severe in the control group and the L-VR group. It was milder in the H-VR group evidently contrary to the rice vinegar group as shown in Fig. 1. Liver cell steatosis was severe in the control group, the L-VR group and the rice vinegar group, but it was milder in the H-VR group. Necrosis of liver cells was mild in the control group, but it was not obvious in the others. The lymphocytes infiltrate in hepatic lobule was not obvious in all three treatment groups.

Fig. 1 Effects of orally administered rice vinegar residue and rice vinegar on liver damage

2.4 Morphological analysis of tissue in hamsters

Morphological parameters of adipocytes in hamsters were shown in the 4 groups (Fig. 2). The numbers of complete cells were counted in the same field of vision to explain the cell sizes. The larger the cell number is, the smaller the cell size will be. Compared with that of the control group, average adipocyte size of mesenteric adipocytes in the L-VR group was significantly smaller (P < 0.05).Average adipocyte size in the H-VR and rice vinegar groups was smaller than that in the L-VR group (P < 0.05). As for the epididymal adipocytes, both rice vinegar residue and rice vinegar groups were smaller than that of the control group(P < 0.05). The perirenal adipocytes sizes in the L-VR and rice vinegar groups were smaller than that in the control group, but larger than that in the H-VR group (P < 0.05).

Fig. 2 Effects of orally administered rice vinegar residue and rice vinegar on adipocyte size in white adipose tissue

2.5 Physiological activity of rice vinegar residue in vitro

Fig. 3 Effects of rice vinegar residue and rice vinegar on intestinal digestion and absorption of cholesterol and triglyceride in vitro

Fig. 3A showed the change of in vitro cholesterol concentration in cholate micelles. The cholesterol concentration in the micelle was decreased gradually(P < 0.05) with the increase of samples concentration. Inhibition effect of the rice vinegar was more obvious than that of the rice vinegar residue at the same sample concentration (P < 0.05).Fig. 3B showed the change of cholate concentration in supernatant. The cholate concentration in supernatant was decreased with the increase of the rice vinegar residue concentration in a linear relationship (P < 0.05), while there was no effect of the rice vinegar. Moreover, the effects of the rice vinegar residue and rice vinegar on the pancreatic lipase activity in a dose-dependent manner were also analyzed, but no significant effect was observed (Fig. 3C).

3 Discussion

It has been widely reported that many kinds of vinegars effectively lower the concentrations of plasma TC and LDL-C and reduce the risk of developing hypercholesterolemic subjects and atherosclerosis in animal models[13-14]. In the present study, the rice vinegar residue, both in L-VR and H-VR groups, not only reduced the plasma LDL-C concentrations as the same as the rice vinegar group, but also increased the plasma HDL-C concentrations (Table 2).The effects of rice vinegar residue on lipids metabolism are mainly due to its high contents of fermented dietary fiber[15-16]and rice bran oil[17-18](Table 1), as the reported beneficial effects of these ingredients previously. Therefore,we speculate that the hypocholesterolemic properties of rice vinegar residue did not attribute to one single functional component, but these components together, including dietary fiber, proteins, or lipids.

Liver is the main organ of lipid metabolism, including lipid digestion and absorption, transport, catabolism and anabolism. There will be an imbalance between catabolism and anabolism of liver lipids if the concentrations of plasma lipids are disordered, and lipids will accumulate in liver,which is likely to induce fatty liver[19]. A previous report demonstrated the effect of rice bran oil on regulation of liver TG[18]. Our results showed that besides the decrease of plasma TC and LDL-C, the plasma TG concentrations were lowered significantly in the H-VR and L-VR groups, which might be directly related to the drop of liver TG concentrations(Table 2). The result of liver slices showed that three treatment groups ameliorated hepatic sinus swelling and lymphocytes infiltration (Fig. 1). This result demonstrates that rice vinegar residue treatments in H-VR group also ameliorated necrosis of liver cells by reducing TG deposition,although the molecular mechanism needs further study.

In the present study, the average sizes of adipocytes were significantly smaller in the mesenteric, epididymal and perirenal adipose tissues in three treatment groups (Fig. 2).It has been reported that the decrease of serum cholesterol caused the decrease of adipocyte size due to reduction of leptin in the serum level[20]. Decrease of adipocyte size is meaningful for reducing obesity because the enlargement of adipose tissue results in hyperplasia so as to cause adiposis[21]. The increase of tissue weight of obese mice is characterized by an increase in adipocyte size up to maximum[22]. These results indicated that rice vinegar residue, the same as rice vinegar, may play an important role in slowing down the curse of obesity.

Moreover, the rice bran oil exerted helpful effect on cholesterol metabolism by up-regulation of LDL-receptor expression, which led to increase fecal cholesterol and BAs excretions[23]. It was also reported that more than half of the cholesterol is de novo synthesized in the liver[24]. That means the hypercholesteremia results from both cholesterol absorption and cholesterol synthesis in liver. In this study,the concentrations of TC, TG and BAs in the H-VR and rice vinegar groups were significantly increased and TC concentration was significantly increased in L-VR group.Furthermore, there were also some effects on liver cholesterol even though they were not statistically significant (Table 2).This result indicated that the rice vinegar residue might contribute to the decreased plasma cholesterol level not only by inhibiting its absorption in the intestine and increasing the excretion of BAs, but also inhibiting its synthesis in the liver.

Bioactive substances, such as sitosterols, polypeptides and polysaccharides, lower down the plasma LDL-C concentrations by disrupting intestinal cholesterol solubilization into micelles, which is a necessary step in the efficient absorption of intestinal cholesterol[25]. Moreover, the certain natural dietary fibers and purified fiber constituents have potential capacities of binding cholate when tested in vitro, which may increase BAs excretion, thereby altering BAs balance, affecting cholesterol intestine absorption and reducing hypercholesteremia[26]. Our result showed that both rice vinegar residue and rice vinegar disrupted intestinal cholesterol solubilization into micelles, and the rice vinegar residue combining with cholate reduced the quantity of uncombined cholate in vitro, which can be reabsorbed into bodies (Fig. 3A and B). In addition, it is well known that dietary lipids are not directly absorbed from the intestine,unless it has been digested by the action of pancreatic lipase[27]. To improve obesity and hyperlipidemia, it may be effective to reduce lipids absorption by lipase inhibition[28].In terms of inhibition of lipase activity, rice vinegar residue had no great contribution (Fig. 3C). These results concluded that rice vinegar residue might reduce adipocyte size and hyperlipidemia by inhibiting lipids absorption in the intestine.

4 Conclusion

The present study evidently indicated that rice vinegar residue reduced the concentrations of plasma LDL-C and TG.It increases plasma HDL-C concentrations and decreases the adipocyte size by preventing the absorption of cholesterol in intestine and promoting excretions of fecal lipids and BAs. This ultimately results from the rice vinegar residue disrupting cholesterol dispersing into micelles and binding with cholate. The effects might be due to its high contents of fermented dietary fiber and bran oil. Keeping in view, the in vivo results it is signposted that rice vinegar residue can be efficiently utilized for making novel high value products to uproot the evil of high cholesterol.